Novel bacillus thuringiensis strains active against lepidopteran and coleopteran pests

ABSTRACT

The invention is related to a novel biologically pure  Bacillus thuringiensis  (B.t.) strains active against lepidopteran and coleopteran pests which produces a bipyramidal crystal consisting essentially of at least two delta-endotoxins having a molecular weight of about 130,000 daltons and a rhomboidal crystal consisting essentially of two delta-endotoxins, each having a molecular weight of about 33,000 daltons, as well as spores, crystals, delta-endotoxins and/or mutants thereof. The invention also relates to insecticidal compositions obtainable therefrom. The invention further relates to methods of using the insecticidal compositions to control an insect pest(s) from the order Lepidoptera and/or Coleoptera. The invention also relates to isolated DNA sequences encoding the delta-endotoxins.

[0001] This application is a continuation-in-part of application Ser.No. 08/264,100, filed Jun. 22, 1994, which is a continuation-in-part ofSer. No. 08/194,651, filed Feb. 9, 1994, which is a continuation-in-partof application Ser. No. 08/166,391, filed Dec. 13, 1993, now abandonedwhich is a continuation-in-part of application Ser. No. 07/991,073,filed Dec. 15, 1992, now abandoned.

1. FIELD OF THE INVENTION

[0002] The invention is related to a novel biologically pure Bacillusthuringiensis (B.t.) strain(s) active against lepidopteran andcoleopteran pests which produces a bipyramidal crystal consistingessentially of at least two delta-endotoxins having a molecular weightof about 130,000 daltons and a rhomboidal crystal consisting essentiallyof two delta-endotoxins, each having a molecular weight of about 33,000daltons, as well as spores, crystals, delta-endotoxins and/or mutantsthereof. The invention also relates to insecticidal compositionsobtainable therefrom. The invention further relates to methods of usingthe insecticidal compositions to control an insect pest(s) from theorder Lepidoptera and/or Coleoptera. The invention also relates toisolated DNA sequences encoding the delta-endotoxins.

2. BACKGROUND OF THE INVENTION

[0003] Every year, significant portions of the world commerciallyimportant agricultural crops, including foods, textiles, and variousdomestic plants are lost to pest infestation, resulting in losses in themillions of dollars. Various strategies have been used in attempting tocontrol such pests.

[0004] One strategy is the use of broad spectrum pesticides, chemicalpesticides with a broad range of activity. However, there are a numberof disadvantages to using such chemical pesticides. Specifically,because of their broad spectrum of activity, these pesticides maydestroy non-target organisms such as beneficial insects and parasites ofdestructive pests. Additionally, these chemical pesticides arefrequently toxic to animals and humans, and targeted pests frequentlydevelop resistance when repeatedly exposed to such substances.

[0005] Another strategy has involved the use of biopesticides, whichmake use of naturally occurring pathogens to control insect, fungal andweed infestations of crops. Biopesticides are naturally occuringorganisms that produce a toxin(s), a substance toxic to the infestingagent which is generally less harmful to non-target organisms and theenvironment as a whole than chemical pesticides.

[0006] The most widely used biopesticide is Bacillus thuringiensis(B.t.). B.t. is a widely distributed, rod shaped, aerobic and sporeforming microorganism. During its sporulation cycle, B.t. produces aprotein(s) known as a delta-endotoxin(s), that forms crystallineinclusion bodies within the cell. The delta-endotoxins have molecularweights ranging from 27-140 kD and kill insect larvae upon ingestion.

[0007] Delta-endotoxins have been produced by recombinant DNA methods(see, for example, Tailor et al., 1992, Molecular Microbiology6:1211-1217; toxin is active against lepidopteran and coleopteran pests;Payne et al., U.S. Pat. No. 5,045,469; toxin is active againstlepldopteran pests). The delta-endotoxins produced by recombinant DNAmethods may or may not be in crystal form.

[0008] A number of B.t. strains have been isolated that have been foundto be active against insect pests of the order Lepidoptera. B.t. subsp.kurstaki HD-1 produces bipyramidal and cuboidal crystal proteins in eachcell during sporulation (Lüthy et al., in Microbial and ViralPesticides, ed. E. Kurstak, Marcel Dekker, New York, 1982, pp. 35-74);the bipyramidal crystal was found to be encoded by three czyIA genes(Aronson et al., 1986, Microbiol. Rev. 50:1-50). B.t. subsp. kurstakiHD-73 crystal delta-endotoxin contains the CryIA(c) protein (Adang etal., 1985, Gene 36:289-300). B.t. subsp. dendrolimus HD-7 and HD-37contain a CryIA and a CryII protein; B.t. subsp. sotto contains analkaline soluble protein that differs from the holotype CryIA(a) proteinby 24 amino acids; B.t. subsp. subtoxicus HD-10 contains CryIA and CryIBproteins; B.t. subsp. tolworthi HD-121 contains CryIA and CryIIproteins; and B.t:. subsp. aizawai HD-68 contains CryIA proteins (Höfteand Whiteley, 1989, Microbiol. Reviews 53:242-255). Payne, U.S. Pat. No.4,990,332, issued Feb. 5, 1993, discloses an isolate of B.t., PS85AI,and a mutant of the isolate, PS85AI, which both have activity againstPlutella xylostella, a lepidopteran pest, and produce alkaline solubleproteins having a molecular weight of 130,000 and 60,000 daltons. Payne,U.S. Pat. No. 5,045,469, issued Sep. 3, 1991 discloses a B.t. isolatedesignated PS81F which also produces alkaline soluble proteins having amolecular weight of 130,000 and 60,000 daltons and has activity againstSpodoptera exigua and T. ni; the toxin gene from PS81F appears to havelittle homology to the toxin gene from B.t. subsp. kurstaki HD-1. Payne,U.S. Pat. No. 5,206,166, filed Jun. 25, 1992, issued Apr. 27, 1993,discloses B.t. isolates PS81A2 and PS81RR1 which produce 133,601 and133,367 dalton alkaline-soluble proteins; both have activity againstTrichoplusia ni, Spodoptera exigua and Plutella xylostella and aredifferent from B.t. subsp. kurstaki HD-1 and other B.t. isolate,.Bernier et al., U.S. Pat. No. 5,061,489 and WO 90/03434 discloses strainA20 producing a delta-endotoxin encoded by at least three genes: 6.6-,5.3-, and 4.5-type genes (cryIA(a), cryIA(b), and cryIA(c)). Chestukhinaet al., 1988, FEBS Lett. 232:249-51, disclose that B.t. subsp. galleriaeproduces two delta-endotoxins, both of which are active againstlepidopteran pests.

[0009] Other strains, e.g. Bacillus thuringiensis subsp. tenebrionis(Krieg et al., 1988, U.S. Pat. No. 4,766,203), have been found to bespecific for Coleoptera. The isolation of another coleopteran toxicBacillus thuringiensis strain was reported in 1986 (Hernnstadt et al.Bio/Technology vol. 4, 305-308, 1986, U.S. Pat. No. 4,764,372, 1988).This strain, designated “Bacillus thuringiensis subsp. san diego”, M-7,has been deposited at the Northern Regional Research Laboratory, USAunder accession number NRRL B-15939. However, the assignee of the '372patent, Mycogen, Corp. has publicly acknowledged that Bacillusthuringiensis subsp. san diego is Bacillus thuringiensis subsp.tenebrionis.

[0010] Other isolated strains have been found to be active against twoorders of pests. Padua, 1990, Microbiol. Lett. 66:257-262, discloses theisolation of two mutants containing two delta-endotoxins, a 144 kDprotein having activity against a lepidopteran pest and a 66 kD proteinhaving activity against mosquitoes. Bradfish et al., U.S. Pat. No.5,208,017, discloses B.t. isolates PS86A1 and PS86Q3 which producealkaline soluble proteins having a molecular weight of 58,000 and 45,000daltons and 155,000, 135,000, 98,000, 62,000, and 58,000 daltons,respectively and which have activity against lepidopteran andcoleopteran pests. PCT Application No. WO 90/13651 and Tailor et al.,1992, Molecular Microbiology 6:1211-1217, disclose a B.t. strain whichis toxic against Lepidoptera and Coleoptera and which produces a toxinhaving a molecular weight of 81 kd.

[0011] It is advantageous to isolate new strains of Bacillusthuringiensis to produce new toxins so that there exists a widerspectrum of biopesticides for any giving insect pest.

3. SUMMARY OF THE INVENTION

[0012] The invention is related to a novel biologically pure Bacillusthuringiensis strain(s) or a spore(s), crystal(s) or mutant(s) thereofwhich strain or mutant in contrast to B.t. strains disclosed in theprior art, has activity against an insect pest of the order Lepidopteraand an insect pest of the order Coleoptera, produces at least twodelta-endotoxins having a molecular weight of about 130,000 daltons andtwo delta-endotoxins both having molecular weights of about 33,000daltons. One of the 33,000 dalton delta-endotoxins has an amino acidsequence essentially as depicted in SEQ ID NO:37 (hereinafter referredto as the “MIVDL protein”). The other 33,000 dalton delta-endotoxin hasan amino acid sequence essentially as depicted in SEQ ID NO:38(hereinafter referred co as the “MKHHK protein”). The 130,000delta-endotoxins have insecticidal activity against insect pests of theorder Lepidoptera.

[0013] The invention also relates to each of the delta-endotoxins aswell as an isolated nucleic acid fragment containing a nucleic acidsequence encoding each of the delta-endotoxins or a portion of thedelta-endotoxin having insecticidal activity against a pest. In oneembodiment, the nucleic acid fragment contains a nucleic acid sequenceencoding the MIVDL protein and may have the nucleic acid sequenceessentially as depicted in SEQ ID NO:39. In another embodiment, thenucleic acid fragment contains a nucleic acid sequence encoding theMKHHK protein and may have the nucleic acid sequence essentially asdepicted in SEQ ID NO:40. The invention is also directed to a genomicsequence comprising nucleic acid sequence encoding the MKHHK and/orMIVDL and may have the nucleic acid sequence essentially as depicted inSEQ ID NOS:41 (MKHHK and MIVDL), 44 (MKHHK), 45 (MJVOL)

[0014] The invention also provides vectors, DNA constructs andrecombinant host cells comprising the claimed nucleic acid fragment(s),which vectors, DNA constructs and recombinant host cells are useful inthe recombinant production of the delta-endotoxins of the presentinvention. The nucleic acid fragment may be operably linked totranscription and translation signals capable of directing expression ofthe delta-endotoxin in the host cell of choice. Recombinant productionof the delta-endotoxin(s) of the invention is achieved by culturing ahost cell transformed or transfected with the nucleic acid fragment ofthe invention, or progeny thereof, under conditions suitable forexpression of the delta-endotoxin, and recovering the delta-endotoxinfrom the culture.

[0015] The invention is further related to an oligonucleotide probehaving a nucleotide sequence essentially as depicted in SEQ ID NO:20which can be used to detected the MIVDL protein and and oligonucleotideprobe essentially as depicted in SEQ ID NO:21 which can be used todetect the MKHHK protein.

[0016] In a specific embodiment of the invention, the thuringiensisstrain of the present invention is EMCC0075 and EMCC0076 having theidentifying characteristics of NRRL B-21019 and NRRL B-21020respectively.

[0017] The novel Bacillus thuringiensis strains, spores, mutants orcrystals and/or delta-endotoxins may within the scope of this inventioneach be formulated into insecticidal compositions. In one embodiment,the strain, spores, mutants, crystals, and/or delta-endotoxins may becombined with an insecticidal carrier. Insecticidal compositionscomprising the strains or mutants of the invention and/or spores, and/orcrystals thereof may be used to control insect pests of the orderLepidoptera and and/or insect pests of the order Coleoptera in a methodcomprising exposing the pest to an insect-controlling effective amountof such an insecticidal composition.

[0018] Furthermore, the compositions or delta-endotoxins of the presentinvention may be used to enhance the insecticidal activity of anotherBacillus-related insecticide. As defined herein, “a Bacillus relatedinsecticide” is a Bacillus (e.g., Bacillus thuringiensis, specifically,Bacillus thuringiensis subsp. kurstaki or Bacillus thuringiensis subsp.tenebrionis or Bacillus subtilis) strain, spore, or substance, e.g.,protein or fragment thereof having activity against or which killinsects; a substance that provides plant protection, e.g. antifeedingsubstance; or a microorganism capable of expressing a Bacillus geneencoding a Bacillus protein or fragment thereof having activity againstor which kills insects (e.g., Bacillus thuringiensis delta-endotoxin)and an acceptable carrier (see Section 5.2., infra, for examples of suchcarriers). A microorganism capable of expressing a Bacillus geneencoding a Bacillus protein or fragment thereof having activity againstor which kill insects inhabits the phylloplane (the surface of the plantleaves), and/or the rhizosphere (the soil surrounding plant roots),and/or aquatic environments, and is capable or successfully competing inthe particular environment (crop and other insect habitats) with thewild-type microorganisms and provide for the stable maintenance andexpression of a Bacillus gene encoding a Bacillus protein or fragmentthereof having activity against or which kill insects. Examples of suchmicroorganisms include but are not limited to bacteria, e.g., generaBacillus, Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas,Streptomyces, Rhizobium, Rhodopseudomonas, Methylochilius,Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter,Leuconostoc, Alcaligenes, and Clostridium; algae, e.g. familiesCyanophyceae, Prochlorophyceae, Rhodophyceae, Dinophyceae,Chrysophyceae, Prymnesiophyceae, Xanthophyceae, Raphidophyceae;Bacillariophyceae, Eustigmatophyceae, Cryptophyceae, Euglenophyceae,Prasinophyceae and Chlorophyceae; and fungi, particularly yeast, e.g.,genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces,Rhodotorula, and Aureobasidium.

[0019] In a specific embodiment, the delta-endotoxins or compositions ofthe present invention may act together with Bacillus-relatedinsecticides in a synergistic fashion. In another embodiment, Bacillusstrains active against insect pests of the order Coleoptera may acttogether in a synergistic fashion with delta-endotoxins, Bacillusstrains or spores thereof active against insect pests of the orderLepidoptera to kill insect pests of the order Coleoptera. In yet anotherembodiment, the delta-endotoxins of the present invention may acttogether in a synergistic fashion.

4. BRIEF DESCRIPTION OF THE FIGURES

[0020]FIG. 1 shows the results of PCR analysis of Bacillus thuringiensisstrains for cryI genes by agarose gel electrophoresis. Lane 1 showsmolecular weight markers (1 kb ladder, BRL-GIBCO). Lanes 2 and 3 showanalysis of strains EMCC0075 and EMCC0076 with cryID oligonucleotideprimers described in FIG. 1. Lanes 4-6 show the analysis of Bacillusthuringiensis subsp. tenebrionis, an unknown Bacillus thuringiensisstrain, and Bacillus thuringiensis subsp. aizawai with cryIDoligonucleotide primers. Bacillus thuringiensis subsp. tenebrioniscontains only the cryIIIA gene; the unknown Bacillus thuringiensisstrain does not contain the cryID gene; and Bacillus thuringiensissubsp. aizawai contains several cryI genes including cryID.

[0021]FIG. 2 shows the cloned DNA fragments which encode the MKHHK andMIVDL proteins.

[0022]FIGS. 3A and 3B shows the homology of the “MIVDL” protein to the34 kDa protein of Bacillus thuringiensis subsp. thompsoni and theCryIA(a) protein of Bacillus thuringiensis subsp. kurstaki.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1. Obtaining Delta-Endotoxins

[0023] The spores and crystals of the present invention are obtainablefrom the strains of the present invention. The strains of the presentinvention may be cultured using media and fermentation techniques knownin the art (see, for example, Rogoff et al., 1969, J. Invertebrate Path.14:122-129; Dulmage et al., 1971, J. Invertebrate Path. 18:353-358;Dulmage et al., in Microbial Control of Pests and Plant Diseases, H. D.Burges, ed., Academic Press, N.Y., 1980). Upon completion of thefermentation cycle, the crystals and spores can be harvested byseparating B.t. spores and crystals from the fermentation broth by meanswell known in the art, e.g. centrifugation. The spores and crystals arecontained in the pellet.

[0024] As noted in Section 2, supra, crystals consist essentially of adelta-endotoxin(s). The strains of the present invention produce twotypes of crystals. One is a bipyramidal crystal consisting essentiallyof at least two 130,000 dalton delta-endotoxins. The other is abipyramidal crystal consisting essentially of the two 33,000 daltondelta-endotoxins.

[0025] Purification of the crystals or delta-endotoxins can be carriedout by various procedures known in the art, including, but not limitedto, density gradient centrifugation, chromatography (e.g. ion exchange,affinity, hydrophobic and size exclusion), electrophoretic procedures,differential solubility, or any other standard technique for thepurification of proteins.

[0026] The delta-endotoxins may also be obtained from a recombinant DNAexpression system. Specifically, DNA encoding each toxin as, forexample, essentially depicted in SEQ ID NOS:39, 40, 44, and 45 is clonedinto a suitable DNA expression vector. Alternatively one genomic DNAfragment comprising nucleic acid sequences encoding each delta endotoxinas, for example, essentially depicted in SEQ ID NO:41 may be cloned.

[0027] Identification of the specific DNA fragment encoding thedelta-endotoxin may be accomplished in a number of ways, including, butnot limited to, electrophoretic separation of the fragments (Southern,1975, J. Mol. Biol. 98:503) in agarose, transfer of the separated DNAfragments to nitrocellulose, nylon, or other suitable support medium,and probing of the transferred fragments with a degenerateoligonucleotide probe(s) based on the amino acid sequence of the proteinas determined by sequential Edman degradation. Alternatively, one mayprobe with a labeled gene fragment corresponding to the open readingframe of a protein with suspected high homology to the protein ofinterest. High homology to the gene of interest may be determined byalignment of a family of related proteins and identification of highlyconserved regions in the encoding DNA segments (see, for example,Gribskov, K., and J. Devereux, eds., in Sequence Analysis Primer,Stockton Press, N.Y., 1991). An elegant and reliable method is todetermine the amino acid sequences of at least two peptide fragments,generated by enzymatic or chemical means from the protein of interest,design degenerate oligonucleotides that will recognize the DNA encodingthose regions, and then to apply polymerase chain reaction (PCR)techniques to amplify perfect or near-perfect copies of the interveningregion of DNA. This PCR-generated segment of DNA can then be labeled andused as a highly specific probe for cloning the delta-endotoxin-encodinggene.

[0028] Once identified, the DNA fragment harboring the gene encoding thedelta-endotoxin or a portion thereof may be cloned by ligation of asize-selected library of fragments expected to harbor the gene ofinterest into a suitable vector. including, but not limited to, pBR322,pUC118, pACYC194, and pBCSK plasmids and their variants fortransformation into Escherichia coli; or pUB110, pBD64, pBC16, pHP13,pE194, pC194, and their variants, for transformation into Bacillus spp.Bacteriophage vectors, such as lambda and its derivatives, may also beused for cloning of the gene(s) into E. coli.

[0029] Production of the delta-endotoxin or a portion thereof atcommercially useful levels can be achieved by subcloning the encodinggene into plasmid vectors that permit stable expression and maintenancein a suitable host. Frequently, acceptable expression can be achievedusing the native regulatory elements present on the DNA fragmentencoding the delta-endotoxin. However, one might wish to add or altertranscriptional regulatory signals (promoters, initiation start sites,operators, activator regions, terminators) and translational regulatorysignals (ribosomal binding sites, initiation codons) for enhanced ormore regulated expression of the delta-endotoxin gene within the chosenhost cell.

[0030] In addition to plasmids, delta-endotoxin genes and theappropriate regulatory elements may be introduced into one of the nativeplasmids of Bacillus thuringiensis and/or other chosen host, or into thechromosomal DNA, via “gene conversion” (e.g., Iglesias and Trautner,1983, Mol Gen. Genet. 189:73-76; Duncan et al., 1978, Proc. Natl. Acad.Sci. U.S.A. 75:3664-3665) or homologous recombination (e.g., Ferrari etal., 1983, J. Bacteriol. 154:1513-1515) at sites of shared DNA homologybetween the vector and the host strain. An efficient “two-plasmid”system may be used for introduction of genes into Bacilli via homologousrecombination (see, for example, PCT Patent WO91/09129). Transposons mayalso be used to introduce cry genes into the selected host strain. Forexample, in the Bacilli, transposons such as Tn917 and its derivativesmay be used (Youngman et al., 1989, in Regulation of ProkaryoticDevelopment, I. Smith, R. Slepecky, and P. Setlow, eds American Societyfor Microbiology, Washington, D.C.).

[0031] Transfer of cloned delta-endotoxin genes into Bacillusthuringiensis, as well as into other organisms, may be achieved by avariety of techniques, including, but not limited to, protoplasting ofcells (Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115; Crawford etal., 1987, J. Bacteriol. 169: 5423-5428); electroporation (e.g.,Schurter et al., 1989, Mol. Gen. Genet. 218: 177-181 and Macaluso etal., 1991, J. Bacteriol. 173: 1353-1356); particle bombardment (e.g.,Shark et al., 1991, Appl. Environ. Microbiol. 57:480-485); siliconcarbide fiber-mediated transformation of cells (Kaeppler et al., 1992,Theor. Appl. Genet. 84:560-566); conjugation (Gonzalez et al., 1982,Proc. Natl. Acad. Sci. U.S.A. 79:6951-6955); or transduction bybacteriophage (e.g., Lecadet et al., 1992, Appl. Environ. Microbiol. 58:840-849). Transformed colonies may be detected by their ability toproduce crystal delta-endotoxin, to bind antibody directed against thatspecific delta-endotoxin, or to kill susceptible pests, e.g., arthropodsor nematodes, in bioassay.

[0032] Criteria for selection of a particular host for productioninclude, but are not limited to, ease of introducing the gene into thehost, availability of expression systems, and stable maintenance andexpression of the gene encoding the delta-endotoxin. The host may be amicroorganism, such as Bacillus thuringiensis itself, or an inhabitantof the phytosphere, e.g., the phylloplane (the surface of plants),and/or the rhizosphere (the soil surrounding plant roots), and/oraquatic environments, and should be capable of competing in theparticular environment (crop and other insect habitats) with thewild-type microorganisms. Examples of such microorganisms include butare not limited to bacteria, e.g. genera Bacillus, Pseudomonas, Erwinia,Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Arthrobacter, Azotobacter, Leuconostoc, Alcaligenes,and Methylophilius, Agrobacterium, Acetobacter, Lactobacillus,Clostridium; algae, e.g. families Cyanophyceae, Prochlorophyceae,Rhodophyceae, Dinophyceae, Chrysophyceae, Prymnesiophyceae,Xanthophyceae, Raphidophyceae, Bacillariophyceae, Eustigmatophyceae,Cryptophyceae, Euglenophyceae, Prasinophyceae, and Chlorophyceae; andfungi, particularly yeast, e.g. genera Saccharomyces, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium.

[0033] The gene(s) encoding the delta-endotoxin(s) of the presentinvention or a portion thereof can also be inserted into an appropriatecloning vector for subsequent introduction into the genomes of suitableplants that are known to be infested with insects susceptible to thedelta-endotoxin(s), or into specific baculoviruses which can in turn bedirectly used as insecticides.

[0034] Those-skilled in the art will recognize that the invention is notlimited to use of the nucleic acid fragments specifically disclosedherein, for example, in SEQ ID NO:39 OR 40. It will be apparent that theinvention also encompasses those nucleotide sequences that encode thesame amino acid sequences as depicted in SEQ ID NO:39 OR 40, but whichdiffer from those specifically depicted nucleotide sequences by virtueof the degeneracy of the genetic code. The invention specificallyencompasses any variant nucleotide sequence, and the protein encodedthereby, which protein retains at least about an 80%, preferably 90%,and most preferably 95% homology or identity with one or the other ofthe amino acid sequences depicted in FIG. 2 and retains the activity ofthe sequences described herein. In particular, variants which retain ahigh level (i.e., >80%) of homology at highly conserved regions of saiddelta-endotoxin are contemplated. Furthermore, the invention encompassesany variant that hybridizes to the nucleotide sequence of thedelta-endotoxin under the following conditions: presoaking in 5×SSC andprehydbridizing for 1 hr at about 40° C. in a solution of 20% formamide,5×Denhardt's solution, 50 meet sodium phosphate, pH 6.8, and 50 ugdenatured sonicated calf thymus DNA, followed by hybridization in thesame solution supplemented with 100 uM ATP for 18 hrs. at about 40° C.,followed by a wash in 0.4×SSC at a temperature of about 45° C.

[0035] Useful variants within the categories defined above include, forexample, ones in which conservative amino acid substitutions have beenmade, which substitutions do not significantly affect the activity ofthe protein. By conservative substitution is meant that amino acids ofthe same class may be substituted by any other of that class. Forexample, the nonpolar aliphatic residues Ala, Val, Leu, and Ile may beinterchanged, as may be the basic residues Lys and Arg, or the acidicresidues Asp and Glu. Similarly, Ser and Thr are conservativesubstitutions for each other, as are Asn and Gln. It will be apparent tothe skilled artisan that such substitutions can be made outside theregions critical to the function of the molecule and still result in anactive delta-endotoxin. Retention of the desired activity can readily bedetermined by using the assay procedures described below.

5.2. Mutants

[0036] The invention is also directed to a mutant B.t. strain whichproduces a larger amount of and/or larger crystals than the parentalstrain. A “parental strain” as defined herein is she original Bacillusthuringiensis strain before mutagenesis.

[0037] To obtain such mutants, the parental strain may, for example, betreated with a mutagen by chemical means such asN-methyl-N′-nitro-N-nitrosoguanidine or ethyl methanesulfonate, or byirradiation with gamma rays, X-rays or UV. Specifically, in one methodof mutating Bacillus thuringiensis strains aid selecting such mutantsthe following procedure is used:

[0038] i) the parental strain is treated with a mutagen;

[0039] ii) the thus presumptive mutants are grown in a medium suitablefor the selection of a mutant strain; and

[0040] iii) the mutant strain is selected for increased production ofdelta-endotoxin.

[0041] According to a preferred embodiment of this method, the selectedcolonies are grown in a production medium, and a final selection forstrains capable of increased delta-endotoxin production is performed.

[0042] Alternatively, the mutant(s) may be obtained using recombinantDNA methods known in the art. For example, a DNA sequence containing agene coding for a delta-endotoxin may be inserted into an appropriateexpression vector and subsequently introduced into the parental strainusing procedures known in the art. Alternatively, a DNA sequencecontaining a gene coding for a delta-endotoxin may be inserted into anappropriate vector for recombination into the genome and subsequentamplification.

5.3. Bioassay

[0043] The activity of the B.t. strains of the present invention orspores, mutants, crystals, or delta-endotoxins thereof against variousinsect pests may be assayed using procedures known in the art, such asan artificial insect diet incorporation assay, artificial diet overlay,leaf painting, leaf dip, and foliar spray. Specific examples of suchassays are given in Section 6, infra.

5.4. Compositions

[0044] The strains, spores, crystals, delta-endotoxins, or mutants ofthe present invention described supra can be formulated with anacceptable carrier into an insecticidal composition(s) that is, forexample, a suspension, a solution, an emulsion, a dusting powder, adispersible granule, a wettable powder, an emulsifiable concentrate, anaerosol or impregnated granule.

[0045] Such compositions disclosed above may be obtained by the additionof a surface active agent, an inert carrier, a preservative, ahumectant, a feeding stimulant, an attractant, an encapsulating agent, abinder, an emulsifier, a dye, a U.V. protectant, a buffer, a flow agent,or other component to facilitate product handling and application forparticular target pests.

[0046] Suitable surface-active agents include but are not limited toanionic compounds such as a carboxylate, for example, a metalcarboxylate of a long chain fatty acid; an N-acylsarcosinate; mono ordi-esters of phosphoric acid with fatty alcohol ethoxylates or salts ofsuch esters; fatty alcohol sulphates such as sodium dodecyl sulphate,sodium octadecyl sulphate or sodium cetyl sulphate; ethoxylated fattyalcohol sulphates; ethoxylated alkylphenol sulphates; ligninsulphonates; petroleum sulphonates; alkyl aryl sulphonates such asalkyl-benzene sulphonates or lower alkylnaphthalene sulphonates, e.g.butyl-naphthalene sulphonate; salts of sulphonatednaphthalene-formaldehyde condensates; salts of sulphonatedphenol-formaldehyde condensates; or store complex sulphonates such asthe amide sulphonates, e.g. the sulphonated condensation product ofoleic acid and N-methyl taurine or the dialkyl sulphosuccinates, e.g.the sodium sulphonate or dioctyl succinate. Non-ionic agents includecondensation products of fatty acid esters, fatty alcohols, fatty acidamides or fatty-alkyl- or alkenyl-substituted phenols with ethyleneoxide, fatty esters of polyhydric alcohol ethers, e.g. sorbitan fattyacid esters, condensation products of such esters with ethylene oxide,e.g. polyoxyethylene sorbitar fatty acid esters. block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine as an acetate, naphthenate or oleate;an oxygen-containing amine such as an amine oxide of polyoxyethylenealkylamine; an amide-linked amine prepared by the condensation of acarboxylic acid with a di- or polyamine; or a quaternary ammonium salt.

[0047] Examples of inert materials include but are not limited toinorganic minerals such as kaolin, phyllosilicates, carbonates,sulfates, phosphates or botanical materials such as wood products, cork,powdered corncobs, peanut hulls, rice hulls, and walnut shells.

[0048] The compositions of the present invention can be in a suitableform for direct application or as a concentrate or primary powder whichrequires dilution with a suitable quantity of water or other diluentbefore application. The insecticidal concentration will vary dependingupon the nature of the particular formulation, specifically, whether itis a concentrate or to be used directly. The composition contains 1 to98% of a solid or liquid inert carrier, and 0 to 50%, preferably 0.1 to50% of a surfactant. These compositions will be administered at thelabeled rate for the commercial product, preferably about 0.01 lb-5.0 lbper acre when in dry form and at about 0.01 pts-10 pts per acre when inliquid form.

[0049] In a further embodiment, the strains, spores, crystals,delta-endotoxins or mutants of the present invention can be treatedprior to formulation to prolong the pesticidal activity when applied tothe environment of a target pest as long as the pretreatment is notdeleterious to the crystal delta-endotoxin. Such treatment can be bychemical and/or physical means as long as the treatment does notdeleteriously affect the properties of the composition(s). Examples ofchemical reagents include, but are not limited to, halogenating agents;aldehydes such as formaldehyde and glutaraldehyde; anti-infectives, suchas zephiran chloride; alcohols, such as isopropranol and ethanol; andhistological fixatives, such as Bouin's fixative and Helly's fixative(see, for example, Humason, Animal Tissue Techniques, W. H. Freeman andCo., 1967).

[0050] The compositions of the invention can be applied directly to theplant by, for example, spraying or dusting at the time when the pest hasbegun to appear on the plant or before the appearance of pests as aprotective measure. Plants to be protected within the scope of thepresent invention include, but are not limited to, cereals (wheat,barley, rye, oats, rice, sorghum and related crops), beets (sugar beetand fodder beet), drupes, pomes and soft fruit (apples, pears, plums,peaches, almonds, cherries, strawberries, raspberries, andblackberries), leguminous plants (alfalfa, beans, lentils, peas,soybeans), oil plants (rape, mustard, poppy, olives, sunflowers,coconuts, castor oil plants, cocoa beans, groundnuts), cucumber plants(cucumber, marrows, melons), fibre plants (cotton, flax, hemp, jute),citrus fruit (oranges, lemons, grapefruit, mandarins), vegetables(spinach, lettuce, asparagus, cabbages and other brassicae, carrots,onions, tomatoes, potatoes, paprika), lauraceae (avocados, cinnamon,camphor), deciduous trees and conifers (e.g. linden-trees, yew-trees,oak-trees, alders, poplars, birch-trees, firs, larches, pines), orplants such as maize, turf plants, tobacco, nuts, coffee, sugar cane,tea, vines, hops, bananas and natural rubber plants, as well asornamentals. In most cases, the preferred mode of application is byfoliar spraying. The preferred mode of application for soil pests is byfurrow application or by “lay-by” application. It is generally importantto obtain good control of pests in the early stages of plant growth asthis is the time when the plant can be most severely damaged. The sprayor dust can conveniently contain another pesticide if this is thoughtnecessary. in a preferred embodiment, the compositions of the inventionis applied directly to the plant.

[0051] The compositions of the present invention may be effectiveagainst pests including, but not limited to, pests of the orderLepidoptera, e.g. Achroia grisella, Acleris gloverana, Acleris variana,Adoxophyes orana, Agrotis ipsilon, Alabama argillacea, Alsophilapometaria, Amyelois transitella, Anagasta kuehniella, Anarsialineatella, Anisota senatoria, Antheraea pernyi, Anticarsia gemmatalis,Archips sp., Argyrotaenia sp., Athetis mindara, Bombyx mori, Bucculatrixthurberiella, Cadra cautella, Choristoneura sp., Cochylis hospes, Coliaseurytheme, Corcyra cephalonica, Cydia latiferreanus, Cydia pomonella,Datana integerrima, Dendrolimus sibericus, Desmia funeralis, Diaphaniahyalinata, Diaphania nitidalis, Diatraea grandiosella, Diatraeasaccharalis, Ennomos subsignaria, Eoreuma loftini, Ephestia elutella,Erannis tiliaria, Estigmene acrea, Eulia salubricola, Eupoeciliaambiguella, Euproctis chrysorrhoea, Euxoa messoria, Galleria mellonella,Grapholita molesta, Harrisina americana, Helicoverpa subflexa,Helicoverpa zea, Heliothis virescens, Hemileuca oliviae, Homoeosomaelectellum, Hyphantria cunea, Keiferia lycopersicella, Lambdinafiscellaria fiscellaria, Lambdina fiscellaria lugubrosa, Leucomasalicis, Lobesia botrana, Loxostege sticticalis, Lymantria dispar,Macalla thyrsisalis, Malacosoma sp., Mamestra brassicae, Mamestraconfigurata, Manduca quinquemaculata, Manduca sexta, Maruca testulalis,Melanchra picta, Operophtera brumata, Orgyia sp., Ostrinia nubilalis,Paleacrita vernata, Papilio cresphontes, Pectinophora gossypiella,Phryganidia californica, Phyllonorycter blancardella, Pieris napi,Pieris rapae, Plathypena scabra, Platynota flouendana, Platynotasultana, Platyptilia carduidactyla, Plodia interpunctella, Plutellaxylostella, Rontia protodice, Psendaletia unipuncta, Pseudoplusiaincludens, Sabulodes aegrotata, Schizura concinna, Sitotroga cerealella,Spilonota ocellana, Spodoptera sp., Thaurnstopoea pityocampa, Tineolabisselliella, Trichoplusia ni, Udea rubigalis, Xylomyges curialis,Yponomeuta padella; Coleoptera, e.g., Leptinotarsa sp., Acanthoscelidesobtectus, Callosobruchus chinensis, Epilachna varivestis, Pyrrhaltaluteola, Cylas formicarius elegantulus, Listronotus oregonensis,Sitophilus sp., Cyclocephala borealis, Cyclocephala immaculate,Macrodactylus subspinosus, Popillia japonica, Rhizotrogus majalis,Alphitobius diaperinus, Palorus ratzeburgi, Tenebrio molitor, Tenebrioobscurus, Tribolium castaneum, Tribolium confusum, Tribolius destructor.

[0052] In specific embodiments, a composition comprising the 130,000dalton delta-endotoxins and/or the two 33,000 dalton delta-endotoxins iseffective against lepidopteran pests. Compositions comprising thestrains of the present invention are also effective against lepidopteranand coleopteran pests.

[0053] The following examples are presented by way of illustration, notby way of limitation.

6. EXAMPLES 6.1. Example 1 Cultivating B.t. Strains EMCC0075 AndEMCC0076

[0054] Subcultures of EMCC0075 and EMCC0076, maintained on NutrientBroth Agar slants; are used to inoculate 250 ml baffled shake flaskscontaining 50 ml of medium with the following composition: Corn Steepliquor   15 g/l Maltrin-100   40 g/l Potato Starch   30 g/l KH₂PO₄ 1.77g/l K₂HPO₄ 4.53 g/l

[0055] The pH of the medium is adjusted to 7.0 using 10 N NaOH.

[0056] After inoculation, shake flasks are incubated at 30° C. on arotary shaker with 250 rpm shaking for 72 hours. The B.t. crystals andspores, obtained in the above fermentation, are recovered bycentrifugation at 15,000 rpm for 15 minutes using a Sorvall RC-5Bcentrifuge.

6.2. Example 2 Testing of B.t. Strains EMCC0075 and EMCC0076 Spores andCrystals

[0057] EMCC0075 and EMCC0076 are cultivated in shake flasks as describedin Example 1, supra. To determine if EMCC0075 and EMCC0076 are activeagainst lepidopteran pests, a 1:50 dilution of culture broth is made. 5ml of such diluted culture broth is transferred into a 50 mlpolypropylene centrifuge tube. 20 ml of artificial insect dietcontaining antibiotics is added into the centrifuge tube. The mixture issubsequently dispensed into bioassay trays. Three to six eggs either ofbeet armyworm (Spodoptera exigua) or tobacco budworm (Heliothisvirescens) are applied on the surface of the “diet”. Mylar is ironedonto the bioassay trays and the trays are incubated at 28° C. Scoring iscarried out at 7 and 11 days.

[0058] To determine if EMCC0075 and EMCC0076 are active against insectpests of the order Coleoptera, 5 ml of the culture broths are removedfrom the shake flasks and transferred directly into the 50 mlpolypropylene centrifuge tubes. 20 ml of artificial insect diet(containing known antibiotics) are then added into the tubes (finaltesting concentration=20% w/w) and mixed vigorously. The mixtures arethen dispensed into bioassay trays. Three to six eggs of corn rootworm(Diabrotica undecimpunctata) are applied to the surface of the “diet”.Mylar is ironed onto the bioassay trays and the trays are incubated at28° C. Scoring is carried out at 7 and 11 days.

[0059] The bioactivity of EMCC0075 and EMCC0076 towards Spodopteraexigua and Diabrotica undecimpunctata is expressed in terms of stuntscore (SS). The stunt score is determined after incubating the trays for7 days. In this system, 4=full size larvae (control larvae); 3=¾ size ofcontrol larvae; 2=½ size of control larvae; 1=¼ size of control larvae;and 0=mortality. The smaller the number, the higher the B.t. activity.The results are shown in Table I. It is evident that EMCC0075 andEMCC0076 possess activity against both lepidopteran and coleopteranpests. TABLE I Spodoptera Diabrotica Heliothis exigua undecimpunctatavirescens EMCC0075 1.7 0.9 1.5 EMCC0076 1.8 1.8 1.8 Control 4.0 4.0 4.0

6.3. Example 3 Cry Gene Profile for EMCC0075 and EMCC0076

[0060] The cry gene profile for EMCC0075 and EMCC0076 is determined byusing the PCR method which is described in the Perkin Elmer Cetus GeneAmp® PCR Reagent Kit literature. Double-stranded DNA is heat-denaturedand the two oligonucleotides corresponding to the cryIA(a) gene (listedin the Sequence Listing as SEQ ID NO:3 and SEQ ID NO:4 respectively),cryIA (b) gene (listed in the Sequence Listing as SEQ ID NO:5 and SEQ IDNO:6 respectively), cryIA(c) gene (listed in the Sequence Listing as SEQID NO:7 and SEQ ID NO:8 respectively), cryID gene (listed in theSequence Listing as SEQ ID NO:9 and SEQ ID NO:10 respectively), cryIIIAgene (listed in the Sequence Listing as SEQ ID NO:11 and SEQ ID NO:12respectively), cryIIIB gene (listed in the Sequence Listing as SEQ IDNO:13 and SEQ ID NO:14 respectively), cryIIIC gene (listed in theSequence Listing as SEQ ID NO:15 and SEQ ID NO:16 respectively), andcryIIID gene (listed in the Sequence Listing as SEQ ID NO:17 and SEQ IDNO:18 respectively), are annealed at low temperature and then extendedat an intermediate temperature.

[0061] PCR analysis indicated that both strains contain a cryID-likegene. A probe specific to cryID also detected a cryID-like gene inSouthern analysis of restricted genomic DNA from both strains. No PCRamplifications are observed with primers to cryIA(a), cryIA(b),cryIA(c), cryIB (SEQ ID NOS:22 and 23), cryIC (SEQ ID NOS:24 and 25),cryID, cryIE (SEQ ID NOS:26 and 27), cryIF (SEQ ID NOS:28 and 29), orcyrIG (SEQ ID NOS:30 and 31), nor to cryIIA (SEQ ID NOS:32 and 33),cryIB (SEQ ID NOS:34 and 33), or cryIIC (SEQ ID NOS: 35 and 36), nor tocryIIIA, cryIIIB, cryIIIC, or cryIIID. However, Southern analysis of arestriction fragment from genomic DNA from EMCC0075 and EMCC0076 with aprobe that can detect cryIA(a), cryIA(b), and cryIA(c) confirmed thepresence of a cryIA-like gene.

6.4. Example 4 Purification of EMCC0075 Bipyramidal and RhomboidalCrystals

[0062] A subculture of EMCC0075, maintained on a Nutrient Broth agarplate, is used to inoculate a 2.0 liter baffled shake flask containing500 ml of medium with the same composition as described in Example 5,infra. After inoculation, the shake flask is incubated at 30° C. on arotary shaker for 72 hours at 250 rpm. The crystals and spores arerecovered by centrifugation at 10,000 rpm (Sorvall GSA rotor) for 30minutes. The pellets are washed with deionized water, centrifuged at15,000 rpm (Sorvall SS34 rotor), and resuspended in deionized water bysonication to a concentration of 0.1 g wet weight per ml. 1 g wet weightcrude crystals are diluted to 33.2 ml with deionized water and placed ina 250 ml separatory funnel. The bottom phase solution comprised of 10 ml3M sodium chloride, 23.4 ml 20% polyethylene glycol 8000, and 33.4 ml20% sodium dextran sulfate is added to the 250 ml separatory funnel andmixed, followed by 100 ml of a polyethylene glycol upper phase solutioncomprised of 0.3 g sodium dextran sulfate, 70.3 g polyethylene glycol8000, and 17.5 g sodium chloride per liter deionized water. Thesuspension is shaken vigorously, and the two phases are allowed toseparate at room temperature for 30 minutes.

[0063] The upper phase which contains large quantities of spores isremoved with a pipet. The lower phase contains crystals and residualspores. The extraction is repeated several times until the upper phasecontains essentially no spores. The lower phase is then diluted with 100ml deionized water, and centrifuged at 10,000 rpm (Sorvall GSA rotor)for 45 minutes at 50° C. to recover the crystals. The recovered crystalsare washed with 200 ml deionized water, and recentrifuged as before. Thespores from the upper phase are also recovered using the above washingprocedure.

[0064] The bipyramidal and rhomboidal crystals are then further purifiedby density gradient centrifugation using a discontinuous Ludox™ HS-40(DuPont) gradient comprised of 3.8 ml each of 75%, 50%, and 38% Ludox™v/v adjusted to pH 2.5 with 0.2M Tris-HCl. 10 mg of crystals in 100 μldeionized water are layered on the top of the gradient, and centrifugedin a Beckman Ultracentrifuge at 10,000 rpm (Beckman 41 Ti rotor) for 15minutes at 20° C. Four separate bands are obtained. One contains purerhomboidal crystals and another contains pure bipyramidal crystals. Thetwo other bands contains mixtures of the two crystal types. The purecrystal bands are recovered, washed with deionized water, and used forbioassay.

6.5.Example 5 SDS-PAGE Analysis of the Delta-Endotoxins from EMCC0075and EMCC0076

[0065] Subcultures of EMCC0075 and EMCC0076, maintained on NutrientBroth agar plates, are used to inoculate 250 ml baffled shake flaskscontaining 50 ml of medium with the following composition: Glucose 2.0g/l KH₂PO₄ 0.86 g/l K₂HPO₄ 0.55 g/l Sodium Citrate 2.0 g/l CaCl₂ 0.1 g/lMnCl₂ • 4H₂O 0.16 g/l MgCl₂ • 6H₂O 0.43 g/l ZnCl₂ 0.007 g/l FeCl₃ 0.003g/l Casamino Acids 5 g/l

[0066] After inoculation, the shake flasks are incubated at 30° C. on arotary shaker for 72 hours at 250 rpm. The B.t. crystals obtained in theabove fermentations of EMCC0075 and EMCC0076 are recovered bycentrifugation at 10,000 rpm (Sorvall GSA rotor) for 30 minutes. TheB.t. crystals are then purified by biphasic extraction using sodiumdextran sulfate and polyethylene glycol as outlined in Example 4, supra.

[0067] B.t. crystal preparations from EMCC0075 and EMCC0076 are analyzedby SDS-PAGE. Specifically, the SDS-PAGE is carried out on 10-15%gradient gels using Pharmacia's Phast System™. The protein bands areanalyzed on a Pharmacia densitometer using Pharmacia Gelscan™ Software.The results indicated that the crystals produced by both strains containat least two proteins with molecular weights of approximately 130,000daltons and 33,000 daltons.

6.6. Example 6 Bioassay Using Spodoptera exigua to Determine Activity ofNovel Lepidopteran Active Bacillus thuringiensis Strains

[0068] To determine if purified bipyramidal and rhomboidal crystals areactive against lepidopteran pests, the crystals are bioassayed againstSpodoptera exigua using a surface overlay assay. Samples of crystalpreparations are applied to individual wells of a jelly tray containing500 μl of solidified artificial insect diet per well. The trayscontaining the various samples are air dried. Two to four 2nd or early3rd instar Spodoptera exigua are added to each well containing the driedtest sample. The trays are then sealed with Mylar punched with holes forair exchange and are incubated for 3 days at 300° C. The degree ofstunting, as described in Example 2, supra, is then recorded.

[0069] The results are shown in Table II. It is evident that,surprisingly, both the bipyramidal crystal and the rhomboidal crystalpossess activity against Spodoptera exigua. The spores also showactivity against Spodoptera exigua. TABLE II Sample Wet Weight Stuntscore No crystals or spores — 4  Rhomboidal & bipyramidal 2.5 mg/well 1 crystals and spores 5.0 mg/well 0-1 Both crystals, no spores 2.5 mg/well1  10 mg/well 0-1 Bipyramidal crystals 0.092 mg/well 1  0.48 mg/well 0-1Rhomboidal crystals 0.05 mg/well 1  0.1 mg/well 0-1 0.5 mg/well 0 Spores 10 mg/well 0-1 20 mg/well 0 

6.7. Example 7 Bioassay Against Diabrotica undecimpunctata

[0070] The coleopteran activity of the whole culture broth of EMCC0075,prepared as described in EXAMPLE 1, is bioassayed against Diabroticaundecimpunctata using a micro-diet incorporation bioassay. Specifically,artificial diet is prepared comprised of water, agar, sugar, casein,wheat germ, methyl paraben, sorbic acid, linseed oil, cellulose, salts,propionic acid, phosphoric acid, streptomycin, chlortetracycline, andvitamins. The artificial diet is developed to allow samples consistingof rehydrated dry powders and liquids to be incorporated at a rate of20% v/v. The test sample is prepared in microcentrifuge tubes to yieldeight serial dilutions. The whole broth sample is tested neat at 200μl/ml, and then diluted in 0.1% Tween 20™ to contain 132 μl/ml, 87μl/ml, 66 μl/ml, 44 μl/ml, 30 μl/ml, 20 μl/ml, and 13 μl/ml. The moltenmixture is vortexed and pipetted in 0.1 ml aliquots into 10 wells of a96 well microtiter plate. Control samples containing 0.1% Tween 20™ aredsipensed into 16 wells. Once the diet has cooled and solidified, twoneonate Diabrotica undecimpunctata larvae are added to each well, andthe trays are covered with a perforated sheet of clear mylar. The traysare then incubated for five days at 28±20° C. and 65% relative humidity.

[0071] After five days, insect mortality is rated. The mylar sheet isremoved and each well of the microtiter plate is inspected using adissecting microscope. Larvae that do not move when prodded with adissecting needle are counted as dead. Percent mortality is calculated,and the data is analyzed via parallel probit analysis. The LC₅₀, LC₉₀,slope of regression lines, coefficient of variation (CV), and potenciesare determined.

[0072] The results as shown in Table III indicate the whole culturebroth from EMCC-0075 has a LC₅₀ and a LC₉₀ of 51 μl/ml diet and 170μl/ml diet, respectively, against Diabrotica undecimpunctata. TABLE IIILC₅₀ LC₉₀ μl/ml μl/ml Slope CV N 51 170 2 7 8

6.8. Example 8 Protein Sequencing of the Delta-Endotoxins from theRhomboidal Crystal Proteins of EMCC0075

[0073] 60 μl of 50% trifluoroacetic acid (TFA) are added to 25 μg ofrhomboidal crystals. Four 15 μl aliquots of the mixture are spot driedonto a Biobrene-coated and TFA-pretreated microcartridge glass fiberfilter. N-terminal sequencing is performed on a Applied Biosystems Inc.Protein Sequencer Model 476A with on-line HPLC and liquid phase TFAdelivery. HPLC determination of phenylthiohydantoin-amino acids isachieved by using the Premix buffer system (ABI Inc.). Data is collectedon a Macintosh IIsi using ABI's 610 data analysis software.

[0074] A double sequence is observed at approximately a 60/40 ratio.Data are analyzed and the sequences are sorted as follows:

[0075] “MIVDL”: MIVDLYRYLGGLAAVNAVLHFYEPRP (SEQ ID NO:1)

[0076] “MKHHK”: MKHHKNFDHI (SEQ ID NO:2)

6.9. Example 9 Cloning of the Genes Encoding the “MIVDL” and “MKHHK”Proteins”

[0077] The amino acid sequence initially determined for the “MIVDL”protein, MIVDLYRYLGGLAAVNAVLHFYEPRP, is encoded by the sequence ATG ATHGTN GAY YTN TAY MGN TAY YTN GGN GGN YTN GCN GCN GTN AAY GCN GTN YTN CAYTTY TAY GAR CCN MGN CCN (SEQ ID NO:19). Based on this sequence, a 71 ntoligomer is designed, where mixed deoxynucleotides are used at the2-fold redundant positions and deoxyinosine at the 4-fold redundantpositions to decrease both base discrimination at mismatches andselectivity at incorrect bases (Martin, F. H., and M. M. Castro, 1985,Nucleic Acids Res. 13: 892-8938): ATG ATI GTI GAY YTI TAY MGI TAY YTIGGI GGI YTI GCI GCI GTI AAY GCI GTI YTI CAY TTY TAY GAR CC (SEQ IDNO:20).

[0078] The amino acid sequence determined for the “MKHHK” protein,namely, MKHHKNFDHI, permitted design of a more discriminating probebecause of the absence of amino acids specified by more than two codons.Further discrimination is permitted by the assumption that As or Tswould be used in the coding-sequence in preference to Gs or Cs, due tothe overall low % G+C content of B.t. strains (approx 34 moles %, Claus,D., and R. C. W. Berkeley. 1986. Genus Bacillus, p. 1112. In P. H. A.Sneath (ed.), Bergey's manual of systematic bacteriology, v. 2. TheWilliams and Wilkins Co., Baltimore). The following probe issynthesized: ATG AAA CAT AAA AAT TTT GAT CAT AT (SEQ ID NO:21). Both theMIVDL and the MKHHEK probes are tailed with digoxygenin-dUTP accordingto the manufacturer's instructions (Boerhinger-Mannheim Genius System™Users Guide, version 2.0).

[0079] EMCC0075 genomic DNA is digested with EcoRI, EcoRV, HindIII,PstI, or combinations of those enzymes overnight in buffers supplied bythe manufacturers, electrophoresed through 0.8% agarose in 0.5×TBE(TRIS-borate-EDTA buffer; Sambrook et al., 1989, in Molecular Cloning, aLaboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor,N.Y.), transferred in 10×SSC to Boehringer Mannheim nylon membrane witha Stratagene Posiblotter in 10×SSC, and then probed as described below.The MIVDL probe, after hybridization and stringent washing at 480° C.with 0.5×SSC, detected EcoRV and PstI fragments 12 kb or more in size,an EcoRI fragment of approx 10 kb, and a HindIII fragment of approx 3.5kb. The MKHHK probe, after hybridization and stringent washing at 480°C. with 5×SSC, detected the same size EcoRI, EcoRV, and PstI fragmentsas did the MIVDL probe. This result indicates that the two genes lie inclose proximity to each other. Additionally, the MKHHK probe detected aHindIII fragment of approx 6 kb.

[0080] To clone the HindIII fragments encoding at least part of the“MIVDL” and “MKHHK” proteins, pUC118 is digested with HindIII, and thentreated with calf intestinal phosphatase to dephosphorylate the 5′ endsand thus prevent vector religation. Restricted and phosphatased pUC118is then mixed with EMCC0075 genomic DNA that had been previouslydigested to completion with HindIII. After ligation, the reaction mix isused to transform E. coli strain XL1-Blue MRF' (Stratagene, Inc., LaJolla, Calif.). Colonies harboring the desired DNA fragment are detectedby “colony hybridization” with the aforementioned “MIVDL” and “MKHHK”probes by the procedure described by Sambrook et al., 1989, Molecularcloning, A Laboratory Manual, Cold Spring Laboratory Press, Cold SpringHarbor, N.Y. Three fragments are cloned with the “MIVDL” and “MKHHK”probes (see FIG. 2). E. coli containing the “13D” MIVDL gene fragmentaew referred to as EMCC0117 cells; E. coli containing the “8D-1” MKHHKgene fragment are referred to as EMCC0118 cells; E. Coli containing the“2B” fragment of the MIVDL and MKHHK genes are referred to as EMCC0118cells.

6.10. Example 10 Sequencing of the Genes Proteins

[0081] Nested deletions of three cloned fragments described in EXAMPLE 9are performed according to the method of Henikoff (Gene 28:351-359,1984) with a Promega “Erase-a-Base” kit. Nested deletion setsencompassing the region of interest are sequenced by the dideoxy method(Sanger et al., 1977, PNAS USA 74:5463-5467) with an ABI 373A sequencer.Sequence correction is performed with SeqEd v 1.0.3; sequence isassembled with MacVector 4.1.1 and AssemblyLIGN v 1.0.7; and additionalalignments and searches are performed with the IntelliGenetics SuitePrograms, v 5.4.

[0082] The determined nucleotide (nt) sequence encoding the MKHHK andMIVDL proteins are shown in SEQ ID NO:39 and 40. The deduced amino acidsequence of the MKHHK and MIVDL proteins is shown underneath theircorresponding DNA sequence. The amino acid sequence determined byN-terminal Edman degradation as described in EXAMPLE 8 is in completeagreement with the sequences deduced from the nucleotide sequence. Thegenomic DNA sequence is shown in SEQ ID NOS:41 (MKHHK and MIVDL), 44(MKHHK), and 45 (MIVDL).

[0083] The MKHHK and MIVDL genes encode proteins with calculatedmolecular masses of 32,719 and 32,866 daltons. The MKHHK protein alignspoorly with any deduced protein from the EMBL, GeneSeq, or GenBanksequence databases. The MIVDL protein has weak regional homology withthe 34 kdal gene of B. thuringiensis subsp. thompsoni as shown in FIG. 3(SEQ ID NO:42) (Brown and Whiteley, 1990, J. Bacteriology 174:549-557).In addition, the MIVDL protein has weak regional homologies withCryIA(a) (SEQ ID NO:43) (see FIG. 3). These weak homologies do notcorrespond to the any of the 5 conserved blocks of Cry toxins describedby Höfte and Whiteley (Microbiol. Rev. 53:242-255, 1989).

[0084] A nucleotide analysis of the region encoding the MKHHK and MIVDLgenes shows ribosome binding sites (AAGGAGT and AAGGTGG, respectively)that differ by one nucleotide with the canonical ribosome binding siteof B. subtilis (AAGGAGG, which is presumably similar to the B.thuringiensis RBS). There is a reasonable transcriptional terminatordownstream of the MIVDL gene.

7. DEPOSIT OF MICROORGANISMS

[0085] The following strains of Bacillus thuringiensis have beendeposited in the Agricultural Research Service Patent Culture CollectionLaboratory (NRRL), Northern Regional Research Center, 1815 UniversityStreet, Peoria, Ill., 61604, USA. Strain Accession Number Deposit DateEMCC0075 NRRL B-21019 Dec. 3, 1992 EMCC0076 NRRL B-21020 Dec. 3, 1992

[0086] The strains have been deposited under conditions that assure thataccess to the culture will be available during the pendency of thispatent application to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122 and under conditions of the Budapest Treaty. The deposit representsa biologically pure culture of each deposited strain. The deposit isavailable as required by foreign patent laws in countries whereincounterparts of the subject application, or its progeny are filed.However, it should be understood that the availability of a deposit doesnot constitute a license to practice the subject invention in derogationof patent rights granted by governmental action.

[0087] The invention described and claimed herein is not to be limitedin scope by the specific embodiments herein disclosed, since theseembodiments are intended as illustrations of several aspects of theinvention. Any equivalent embodiments are intended to be within thescope of this invention. Indeed, various modifications of the inventionin addition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are also intended to fall within the scope of the appendedclaims.

[0088] Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1 45 26 amino acids amino acid single linear peptide 1 Met Ile Val AspLeu Tyr Arg Tyr Leu Gly Gly Leu Ala Ala Val Asn 1 5 10 15 Ala Val LeuHis Phe Tyr Glu Pro Arg Pro 20 25 10 amino acids amino acid singlelinear peptide 2 Met Lys His His Lys Asn Phe Asp His Ile 1 5 10 20 basepairs nucleic acid single linear cDNA 3 CTGCTCCAGC TGCTTGGCTC 20 22 basepairs nucleic acid single linear cDNA 4 GAATTATACT TGGTTCAGGC CC 22 22base pairs nucleic acid single linear cDNA 5 GCACACCTTA CATTTTAAAG CA 2227 base pairs nucleic acid single linear cDNA 6 AGATTACAAG CGGATACCAACATCGCG 27 21 base pairs nucleic acid single linear cDNA 7 TGGCACTTTCAAAATAACCA A 21 26 base pairs nucleic acid single linear cDNA 8GCATCGGATA GTATTACTCA AATCCC 26 22 base pairs nucleic acid single linearcDNA 9 CGCTCTAACA TAGACCTTAT AA 22 26 base pairs nucleic acid singlelinear cDNA 10 GACATTTCAT TAGGGCTTAT TAATTT 26 22 base pairs nucleicacid single linear cDNA 11 CAGCGGACGG CCAGACCGCA AG 22 24 base pairsnucleic acid single linear cDNA 12 GTCGGAGTCA ACAACCTTAG GGGC 24 21 basepairs nucleic acid single linear cDNA 13 ATCCGGAAAA GCCGCTATGT C 21 21base pairs nucleic acid single linear cDNA 14 ATCCGGAAAA GCCGCTATGT C 2124 base pairs nucleic acid single linear cDNA 15 GGCCAGAAAA TGGAAAAATTTGGG 24 21 base pairs nucleic acid single linear cDNA 16 GTGGGTACAGGAGGTACCAA A 21 21 base pairs nucleic acid single linear cDNA 17GTGGGTACAG GAGGTACCAA A 21 23 base pairs nucleic acid single linear cDNA18 CGAAATACTA TGAGTGTAAC TGC 23 54 base pairs nucleic acid single linearcDNA 19 YTNGGNGGNY TNGCNGCNGT NAAYGCNGTN YTNCAYTTYT AYGARCCNMG NCCN 5457 base pairs nucleic acid single linear cDNA 20 ATGATGTGAY YTTAYMGTAYYTGGGGYTGC GCGTAAYGCG TYTCAYTTYT AYGARCC 57 29 base pairs nucleic acidsingle linear cDNA 21 ATGAAACATC ATAAAAATTT TGATCATAT 29 31 base pairsnucleic acid single linear cDNA 22 TTGAATTCAT ATCTACTAAT GAGCAATCGA A 3122 base pairs nucleic acid single linear cDNA 23 CCACACGCCT AGATTCTCATGC 22 46 base pairs nucleic acid single linear cDNA 24 CGGGATCCACAGTTACAGTC TGTAGCTCAA TTACCTACTT TTAACG 46 23 base pairs nucleic acidsingle linear cDNA 25 GGCCAAGGTT GCTGTAATAA TCG 23 24 base pairs nucleicacid single linear cDNA 26 CTCAATATTC TCGAAGCTGG GGCC 24 23 base pairsnucleic acid single linear cDNA 27 GCAGTCTGTA CGGAATTTAT ACA 23 22 basepairs nucleic acid single linear cDNA 28 CGAGGGTTAG CAGATAGCTA TG 22 21base pairs nucleic acid single linear cDNA 29 AAGATGGGGC GGTCTAACTC C 2124 base pairs nucleic acid single linear cDNA 30 GACCGTTATC GGGTGAATCTTTAG 24 24 base pairs nucleic acid single linear cDNA 31 TCGGCTGCACTCTAAATTGT TGAG 24 22 base pairs nucleic acid single linear cDNA 32TATTGAGTGA ATTATGGGGG AT 22 23 base pairs nucleic acid single linearcDNA 33 ATGTTCTAAA TTCTAACATA TCG 23 22 base pairs nucleic acid singlelinear cDNA 34 TTATACCTAG ATCCTATTGT TG 22 23 base pairs nucleic acidsingle linear cDNA 35 TAACATTTCC ACACTTTTCA ATC 23 19 base pairs nucleicacid single linear cDNA 36 AAGGCTAGCG ACTGCTGTC 19 287 amino acids aminoacid single linear peptide 37 Met Lys His His Lys Asn Phe Asp His IleVal Trp Asp Phe Ala Glu 1 5 10 15 Lys Trp Thr Glu Gln Lys Gly Val AspLeu Lys Arg Val Ser Tyr Val 20 25 30 Asp Pro Ile Thr Gly Glu Asp Thr LeuGlu Phe Ile Thr Lys Phe Asn 35 40 45 Tyr Val Gly Lys Leu Glu Glu Lys AlaTyr Cys Pro Glu Val Ile Glu 50 55 60 Thr Gln Ser Phe Ser Asn Ser Asn CysAsp Val Ser Arg Glu Phe Leu 65 70 75 80 Lys Lys Lys Val Asp Arg Lys GluCys Tyr Leu Trp Asp Ile Asp Tyr 85 90 95 Gly Phe Ile Ile Pro Thr Ser ValLeu Thr Asn Pro Leu Leu Pro Pro 100 105 110 Thr Leu Asn Glu Lys Ile AsnPro Ala Met Glu Val Asp Leu Phe Lys 115 120 125 Ser Ala Asn Leu Phe GluSer Lys Leu Asn Asn Tyr Arg Met Ile Glu 130 135 140 Ala Gly Val Tyr IleGlu Pro Asn Gln Ala Val Thr Ala Ser Ile Met 145 150 155 160 Val Thr ProLys Gln Val Gln Gln Asp Tyr Cys Ile Ser Leu Glu Ile 165 170 175 Ser GlySer Ile Ile Ile Glu Leu Lys Asp Ala Tyr Asn Ala Cys Thr 180 185 190 AspLys Glu Thr Ile Glu Thr Ile Phe Tyr Thr Val Pro Ile Ala Asp 195 200 205Ile Tyr Arg Ser Glu Leu Ala His Asn His Ser Phe His Leu Asp Gly 210 215220 Glu Thr Val Ile Phe Thr Gly Lys Gly Thr Phe Lys Gly Leu Ile Cys 225230 235 240 Ser Asn Ile Phe Val Glu Gly Glu Arg Phe Asp Ser Gln Thr GlyGlu 245 250 255 Cys Leu Gly Lys Tyr Val Ile Pro Leu Ser Ile Glu Lys LysAsn Asn 260 265 270 Val Asp Cys Ile Ser Ile Phe Leu Asn Ser Glu Lys GlyGly Ile 275 280 285 294 amino acids amino acid single linear peptide 38Met Ile Val Asp Leu Tyr Arg Tyr Leu Gly Gly Leu Ala Ala Val Asn 1 5 1015 Ala Val Leu His Phe Tyr Glu Pro Arg Pro Asp Ile Cys Arg Asn Ile 20 2530 Ser Glu Glu Tyr Asn Leu Ile Val Phe Gly Asp Arg Ile Pro Thr Phe 35 4045 Ser Ile Asp Pro Ser Gln Ile Asn Ile Asn Asn Leu Ser Val Asp Thr 50 5560 Pro Val Asp Glu Ile Thr Ile Asn Asn Val Arg Ser Ile Gln Leu Ile 65 7075 80 Ser Ser Arg Phe Glu Asn Thr Gly Phe Val Asp Thr Glu Asn Tyr Phe 8590 95 Thr Pro Glu Leu Ser Arg Thr Val Val Asn Ser Ile Ser Thr Ser Thr100 105 110 Thr Thr Gly Tyr Lys Tyr Thr Gln Ser Leu Thr Val Ser Ser LysPhe 115 120 125 Ser Phe Asn Phe Pro Val Ala Gly Ala Glu Asn Asn Ile SerPhe Ser 130 135 140 Val Gly Phe Glu Gln Asn Leu Ser Thr Thr Glu Thr LysThr Glu Ser 145 150 155 160 Thr Ser Thr Leu Met Arg Ile Pro Pro Gln ProVal Ser Val Arg Pro 165 170 175 Arg Thr Ala Lys Arg Val Glu Ile Ser LeuPhe Glu Leu Ala Ile Pro 180 185 190 Arg Ile Gln Asn Glu Ile Ser Gly PheVal Thr Gly Thr Leu Pro Thr 195 200 205 Ile Ser Asn Ser His Ile Ser AspLeu Tyr Ala Val Leu Thr Arg Thr 210 215 220 Asp Ser Leu Cys Pro Asn SerTyr Ile Asn Arg Asp Asp Phe Leu Arg 225 230 235 240 Ile Asp His Glu AsnArg Gly Leu Gly Leu Gln Gly Phe Gly Ser Leu 245 250 255 Thr Gly Asn LeuThr Ser Leu Asp Phe Ala Ile Arg Thr Thr Glu Tyr 260 265 270 Asp Leu ProSer Asn Thr Ile Ile Asn Ile Glu Asn Glu Ile Lys Arg 275 280 285 Ala HisIle Leu Thr Gln 290 864 base pairs nucleic acid single linear DNA(genomic) 39 ATGAAACATC ATAAAAATTT TGATCACATA GTTTGGGACT TCGCTGAAAAGTGGACTGAA 60 CAAAAGGGGG TAGATTTAAA AAGGGTCAGT TATGTAGATC CCATTACTGGTGAAGATACA 120 TTAGAGTTTA TAACCAAATT TAATTATGTT GGGAAATTAG AAGAAAAAGCTTATTGTCCA 180 GAAGTAATAG AAACACAATC TTTTTCAAAC TCAAATTGTG ACGTTTCGAGGGAATTTCTA 240 AAGAAAAAAG TAGACAGGAA GGAATGTTAT TTATGGGATA TAGACTATGGGTTTATTATA 300 CCAACTTCGG TACTTACAAA TCCATTATTA CCCCCCACTC TCAATGAAAAAATTAATCCA 360 GCAATGGAAG TGGACTTATT TAAAAGTGCA AACCTGTTTG AATCCAAACTAAATAATTAT 420 AGAATGATAG AAGCAGGTGT TTATATTGAA CCAAATCAAG CAGTAACCGCCAGCATAATG 480 GTTACACCAA AACAAGTACA GCAAGATTAT TGTATTAGCC TTGAGATTTCAGGTAGTATT 540 ATCATTGAGC TGAAAGATGC TTATAATGCT TGTACAGATA AAGAAACTATTGAAACAATA 600 TTCTATACCG TGCCAATTGC AGATATATAC AGATCCGAGC TTGCCCATAACCATTCCTTT 660 CATTTAGATG GAGAAACTGT AATATTTACA GGGAAAGGTA CGTTTAAAGGCTTAATATGT 720 TCTAATATAT TTGTTGAAGG GGAAAGATTC GATTCTCAAA CGGGGGAATGTTTGGGGAAA 780 TATGTGATCC CATTAAGTAT AGAAAAGAAA AATAATGTAG ATTGTATCTCTATATTTTTA 840 AATTCAGAAA AAGGTGGGAT TTAA 864 885 base pairs nucleicacid single linear DNA (genomic) 40 ATGATAGTAG ATTTATATAG ATATTTAGGTGGATTGGCAG CAGTAAATGC CGTACTTCAC 60 TTTTATGAGC CACGCCCTGA TATATGTAGGAATATAAGCG AAGAATATAA CCTTATAGTA 120 TTTGGAGACC GTATACCAAC TTTTAGCATAGATCCTTCGC AAATAAATAT TAACAATTTA 180 TCTGTGGACA CTCCAGTGGA TGAAATAACTATTAATAACG TGAGAAGTAT ACAATTAATA 240 TCTAGTCGTT TTGAAAATAC AGGATTTGTCGATACTGAAA ATTATTTTAC TCCTGAATTA 300 TCTAGAACAG TTGTAAATAG CATATCTACATCGACTACTA CAGGATATAA GTACACTCAA 360 TCCCTTACTG TTTCATCCAA ATTCTCCTTTAATTTCCCAG TTGCGGGTGC AGAAAATAAT 420 ATTTCATTTT CAGTAGGTTT TGAACAAAACCTTTCAACTA CAGAAACTAA AACAGAAAGT 480 ACTTCAACGC TTATGCGTAT ACCTCCACAACCAGTTTCCG TAAGACCCAG AACAGCAAAA 540 AGGGTTGAAA TATCGCTCTT TGAATTGGCAATCCCTAGAA TACAAAACGA AATTTCCGGA 600 TTTGTAACAG GTACTCTTCC AACAATTTCAAATTCGCATA TTTCCGATCT TTATGCTGTA 660 TTAACACGGA CTGATAGCCT ATGCCCTAATTCATATATTA ACCGAGATGA CTTTTTAAGA 720 ATAGATCATG AAAATAGGGG TTTGGGATTACAAGGCTTCG GTTCTCTCAC TGGAAATTTA 780 ACATCATTAG ATTTTGCAAT TAGAACTACTGAATATGATT TACCTTCAAA TACAATTATA 840 AATATAGAGA ACGAAATAAA AAGAGCCCATATACTCACAC AGTAA 885 2101 base pairs nucleic acid single linear DNA(genomic) 41 ATTAAACACT AAATACATTC ACATTATTCT AACAAAGAAA AGGAGTAATAATTATGAAAC 60 ATCATAAAAA TTTTGATCAC ATAGTTTGGG ACTTCGCTGA AAAGTGGACTGAACAAAAGG 120 GGGTAGATTT AAAAAGGGTC AGTTATGTAG ATCCCATTAC TGGTGAAGATACATTAGAGT 180 TTATAACCAA ATTTAATTAT GTTGGGAAAT TAGAAGAAAA AGCTTATTGTCCAGAAGTAA 240 TAGAAACACA ATCTTTTTCA AACTCAAATT GTGACGTTTC GAGGGAATTTCTAAAGAAAA 300 AAGTAGACAG GAAGGAATGT TATTTATGGG ATATAGACTA TGGGTTTATTATACCAACTT 360 CGGTACTTAC AAATCCATTA TTACCCCCCA CTCTCAATGA AAAAATTAATCCAGCAATGG 420 AAGTGGACTT ATTTAAAAGT GCAAACCTGT TTGAATCCAA ACTAAATAATTATAGAATGA 480 TAGAAGCAGG TGTTTATATT GAACCAAATC AAGCAGTAAC CGCCAGCATAATGGTTACAC 540 CAAAACAAGT ACAGCAAGAT TATTGTATTA GCCTTGAGAT TTCAGGTAGTATTATCATTG 600 AGCTGAAAGA TGCTTATAAT GCTTGTACAG ATAAAGAAAC TATTGAAACAATATTCTATA 660 CCGTGCCAAT TGCAGATATA TACAGATCCG AGCTTGCCCA TAACCATTCCTTTCATTTAG 720 ATGGAGAAAC TGTAATATTT ACAGGGAAAG GTACGTTTAA AGGCTTAATATGTTCTAATA 780 TATTTGTTGA AGGGGAAAGA TTCGATTCTC AAACGGGGGA ATGTTTGGGGAAATATGTGA 840 TCCCATTAAG TATAGAAAAG AAAAATAATG TAGATTGTAT CTCTATATTTTTAAATTCAG 900 AAAAAGGTGG GATTTAACAT GATAGTAGAT TTATATAGAT ATTTAGGTGGATTGGCAGCA 960 GTAAATGCCG TACTTCACTT GATTTAAACA TGATAGTAGA TTTATATAGATATTTAGGTG 1020 GATTGGCAGC AGTAAATGCC GTACTTCACT TTTATGAGCC ACGCCCTGATATATGTAGGA 1080 ATATAAGCGA AGAATATAAC CTTATAGTAT TTGGAGACCG TATACCAACTTTTAGCATAG 1140 ATCCTTCGCA AATAAATATT AACAATTTAT CTGTGGACAC TCCAGTGGATGAAATAACTA 1200 TTAATAACGT GAGAAGTATA CAATTAATAT CTAGTCGTTT TGAAAATACAGGATTTGTCG 1260 ATACTGAAAA TTATTTTACT CCTGAATTAT CTAGAACAGT TGTAAATAGCATATCTACAT 1320 CGACTACTAC AGGATATAAG TACACTCAAT CCCTTACTGT TTCATCCAAATTCTCCTTTA 1380 ATTTCCCAGT TGCGGGTGCA GAAAATAATA TTTCATTTTC AGTAGGTTTTGAACAAAACC 1440 TTTCAACTAC AGAAACTAAA ACAGAAAGTA CTTCAACGCT TATGCGTATACCTCCACAAC 1500 CAGTTTCCGT AAGACCCAGA ACAGCAAAAA GGGTTGAAAT ATCGCTCTTTGAATTGGCAA 1560 TCCCTAGAAT ACAAAACGAA ATTTCCGGAT TTGTAACAGG TACTCTTCCAACAATTTCAA 1620 ATTCGCATAT TTCCGATCTT TATGCTGTAT TAACACGGAC TGATAGCCTATGCCCTAATT 1680 CATATATTAA CCGAGATGAC TTTTTAAGAA TAGATCATGA AAATAGGGGTTTGGGATTAC 1740 AAGGCTTCGG TTCTCTCACT GGAAATTTAA CATCATTAGA TTTTGCAATTAGAACTACTG 1800 AATATGATTT ACCTTCAAAT ACAATTATAA ATATAGAGAA CGAAATAAAAAGAGCCCATA 1860 TACTCACACA GTAATTAATA GAAATAGACC GATAATCGGT CTTCCCCCTGTCAAGTAGGC 1920 CTAGTGACAG GGTTCTTGCT GTGGACCGCA AGGTAGCAAA TTTCTGAAGACCCATATGGG 1980 GTACCGTCAG GAAAATGCGG ATTTACAACG CTAAGCCCAT TTTCCTGACGATTCCCCCAT 2040 TTTTAACAAC GTTAAGAAAG TTTCAATGGT CTTAAAGAAT CTAATGAGATCATTTTCTCC 2100 G 2101 310 amino acids amino acid single linear peptide42 Met Ala Ile Met Asn Pro Arg Pro Asp Ile Ala Gln Asp Ala Ala Arg 1 510 15 Ala Trp Asp Ile Ile Ala Gly Pro Phe Ile Arg Pro Gly Thr Thr Pro 2025 30 Thr Asn Arg Gln Leu Phe Asn Tyr Gln Ile Gly Asn Ile Glu Val Glu 3540 45 Thr Pro Pro Gly Asn Leu Asn Phe Ser Val Val Pro Glu Leu Asp Phe 5055 60 Ser Val Ser Gln Asp Leu Phe Asn Asn Thr Ser Val Gln Gln Ser Gln 6570 75 80 Thr Tyr Ala Ser Phe Asn Glu Ser Arg Thr Val Val Glu Thr Thr Ser85 90 95 Thr Ala Val Thr His Gly Val Lys Ser Gly Val Thr Val Ser Ala Ser100 105 110 Ala Lys Phe Asn Ala Lys Ile Leu Val Lys Ser Ile Glu Gln ThrIle 115 120 125 Thr Thr Thr Val Ser Thr Glu Tyr Asn Phe Ser Ser Thr ThrThr Arg 130 135 140 Thr Asn Thr Val Thr Arg Gly Trp Ser Ile Pro Ala GlnPro Val Leu 145 150 155 160 Val Pro Pro His Ser Arg Val Thr Ala Thr LeuGln Ile Tyr Lys Gly 165 170 175 Asp Phe Thr Val Pro Val Leu Gln Asn GluLeu Ser Leu Arg Val Tyr 180 185 190 Gly Gln Thr Gly Thr Leu Pro Ala GlyAsn Pro Ser Phe Pro Ser Asp 195 200 205 Leu Tyr Ala Val Ala Thr Tyr GluAsn Thr Leu Leu Gly Arg Ile Arg 210 215 220 Glu His Ile Ala Pro Pro AlaLeu Phe Arg Ala Ser Asn Ala Tyr Ile 225 230 235 240 Ser Asn Gly Val GlnAla Ile Trp Arg Gly Thr Ala Thr Thr Arg Val 245 250 255 Ser Gln Gly LeuTyr Ser Val Val Arg Ile Asp Glu Arg Pro Leu Ala 260 265 270 Gly Tyr SerGly Glu Thr Arg Thr Glu Tyr Tyr Leu Pro Val Thr Leu 275 280 285 Ser AsnSer Ser Gln Ile Leu Thr Pro Gly Ser Leu Gly Ser Glu Ile 290 295 300 ProIle Ile Asn Pro Val 305 310 358 amino acids amino acid single linearpeptide 43 Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr ValLeu 1 5 10 15 Asp Ile Val Ala Leu Phe Ser Asn Tyr Asp Ser Arg Arg TyrPro Gly 20 25 30 Gly Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr ThrAsn Pro 35 40 45 Val Leu Cys Glu Asn Phe Ser Glu Asp Gly Ser Phe Arg GlyMet Ala 50 55 60 Gln Arg Ile Glu Gln Asn Ile Arg Gln Pro His Leu Met AspIle Leu 65 70 75 80 Asn Ser Ile Thr Ile Tyr Thr Asp Val His Arg Gly PheAsn Tyr Trp 85 90 95 Ser Gly His Gln Ile Thr Ala Ser Pro Val Gly Phe SerGly Pro Glu 100 105 110 Phe Ala Phe Pro Leu Phe Gly Asn Ala Gly Asn AlaAla Pro Pro Val 115 120 125 Leu Val Ser Leu Thr Gly Leu Gly Ile Phe ArgThr Leu Ser Ser Pro 130 135 140 Leu Tyr Arg Tyr Thr Gln Arg Ile Ile LeuGly Ser Gly Pro Asn Asn 145 150 155 160 Gln Glu Leu Phe Val Leu Asp GlyThr Glu Asn Asn Phe Ser Phe Ala 165 170 175 Ser Leu Thr Thr Asn Leu ProSer Thr Ile Tyr Arg Gln Arg Gly Thr 180 185 190 Val Asp Ser Leu Asp ValIle Pro Pro Gln Asp Asn Ser Val Pro Pro 195 200 205 Arg Ala Gly Lys ArgVal Glu Phe Ser Leu His Arg Leu Ser His Val 210 215 220 Thr Met Leu SerGln Ala Ala Gly Ala Val Tyr Thr Leu Arg Ala Pro 225 230 235 240 Thr PheSer Trp Gln His Arg Ser Ala Glu Phe Asn Asn Ile Ile Pro 245 250 255 SerSer Gln Ser Leu Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr Asn 260 265 270Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly Gly 275 280285 Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg Val 290295 300 Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg Tyr305 310 315 320 Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile Asp GlyArg Pro 325 330 335 Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser Ser GlySer Asn Leu 340 345 350 Gln Ser Gly Ser Phe Arg 355 980 base pairsnucleic acid single linear DNA (genomic) 44 ATTAAACACT AAATACATTCACATTATTCT AACAAAGAAA AGGAGTAATA ATTATGAAAC 60 ATCATAAAAA TTTTGATCACATAGTTTGGG ACTTCGCTGA AAAGTGGACT GAACAAAAGG 120 GGGTAGATTT AAAAAGGGTCAGTTATGTAG ATCCCATTAC TGGTGAAGAT ACATTAGAGT 180 TTATAACCAA ATTTAATTATGTTGGGAAAT TAGAAGAAAA AGCTTATTGT CCAGAAGTAA 240 TAGAAACACA ATCTTTTTCAAACTCAAATT GTGACGTTTC GAGGGAATTT CTAAAGAAAA 300 AAGTAGACAG GAAGGAATGTTATTTATGGG ATATAGACTA TGGGTTTATT ATACCAACTT 360 CGGTACTTAC AAATCCATTATTACCCCCCA CTCTCAATGA AAAAATTAAT CCAGCAATGG 420 AAGTGGACTT ATTTAAAAGTGCAAACCTGT TTGAATCCAA ACTAAATAAT TATAGAATGA 480 TAGAAGCAGG TGTTTATATTGAACCAAATC AAGCAGTAAC CGCCAGCATA ATGGTTACAC 540 CAAAACAAGT ACAGCAAGATTATTGTATTA GCCTTGAGAT TTCAGGTAGT ATTATCATTG 600 AGCTGAAAGA TGCTTATAATGCTTGTACAG ATAAAGAAAC TATTGAAACA ATATTCTATA 660 CCGTGCCAAT TGCAGATATATACAGATCCG AGCTTGCCCA TAACCATTCC TTTCATTTAG 720 ATGGAGAAAC TGTAATATTTACAGGGAAAG GTACGTTTAA AGGCTTAATA TGTTCTAATA 780 TATTTGTTGA AGGGGAAAGATTCGATTCTC AAACGGGGGA ATGTTTGGGG AAATATGTGA 840 TCCCATTAAG TATAGAAAAGAAAAATAATG TAGATTGTAT CTCTATATTT TTAAATTCAG 900 AAAAAGGTGG GATTTAACATGATAGTAGAT TTATATAGAT ATTTAGGTGG ATTGGCAGCA 960 GTAAATGCCG TACTTCACTT980 1121 base pairs nucleic acid single linear DNA (genomic) 45GATTTAAACA TGATAGTAGA TTTATATAGA TATTTAGGTG GATTGGCAGC AGTAAATGCC 60GTACTTCACT TTTATGAGCC ACGCCCTGAT ATATGTAGGA ATATAAGCGA AGAATATAAC 120CTTATAGTAT TTGGAGACCG TATACCAACT TTTAGCATAG ATCCTTCGCA AATAAATATT 180AACAATTTAT CTGTGGACAC TCCAGTGGAT GAAATAACTA TTAATAACGT GAGAAGTATA 240CAATTAATAT CTAGTCGTTT TGAAAATACA GGATTTGTCG ATACTGAAAA TTATTTTACT 300CCTGAATTAT CTAGAACAGT TGTAAATAGC ATATCTACAT CGACTACTAC AGGATATAAG 360TACACTCAAT CCCTTACTGT TTCATCCAAA TTCTCCTTTA ATTTCCCAGT TGCGGGTGCA 420GAAAATAATA TTTCATTTTC AGTAGGTTTT GAACAAAACC TTTCAACTAC AGAAACTAAA 480ACAGAAAGTA CTTCAACGCT TATGCGTATA CCTCCACAAC CAGTTTCCGT AAGACCCAGA 540ACAGCAAAAA GGGTTGAAAT ATCGCTCTTT GAATTGGCAA TCCCTAGAAT ACAAAACGAA 600ATTTCCGGAT TTGTAACAGG TACTCTTCCA ACAATTTCAA ATTCGCATAT TTCCGATCTT 660TATGCTGTAT TAACACGGAC TGATAGCCTA TGCCCTAATT CATATATTAA CCGAGATGAC 720TTTTTAAGAA TAGATCATGA AAATAGGGGT TTGGGATTAC AAGGCTTCGG TTCTCTCACT 780GGAAATTTAA CATCATTAGA TTTTGCAATT AGAACTACTG AATATGATTT ACCTTCAAAT 840ACAATTATAA ATATAGAGAA CGAAATAAAA AGAGCCCATA TACTCACACA GTAATTAATA 900GAAATAGACC GATAATCGGT CTTCCCCCTG TCAAGTAGGC CTAGTGACAG GGTTCTTGCT 960GTGGACCGCA AGGTAGCAAA TTTCTGAAGA CCCATATGGG GTACCGTCAG GAAAATGCGG 1020ATTTACAACG CTAAGCCCAT TTTCCTGACG ATTCCCCCAT TTTTAACAAC GTTAAGAAAG 1080TTTCAATGGT CTTAAAGAAT CTAATGAGAT CATTTTCTCC G 1121

What is claimed is:
 1. A biologically pure Bacillus thuringiensis strainhaving insecticidal activity against an insect pest of the orderLepidoptera and an insect pest of the order Coleoptera or spores,crystals or mutants thereof, which strain or mutants produce onedelta-endotoxin having a molecular weight of about 33,000 daltons and anamino acid sequence essentially as depicted in SEQ ID NO:37 and onedelta-endotoxin having a molecular weight of about 33,000 daltons and anamino acid sequence essentially as depicted in SEQ ID NO:38 and at leasttwo delta-endotoxins having a molecular weight of about 130,000 daltonsin which said delta-endotoxins have insecticidal activity against aninsect pest of the order Lepidoptera.
 2. The biologically pure Bacillusthuringiensis strain of claim 1 in which the Bacillus thuringiensisstrain is Bacillus thuringiensis EMCC0075 having the identifyingcharacteristics of NRRL B-21019.
 3. The biologically pure Bacillusthuringiensis strain of claim 1 in which the Bacillus thuringiensisstrain is Bacillus thuringiensis EMCC0076 having the identifyingcharacteristics of NRRL B-21020.
 4. A delta-endotoxin having a molecularweight of about 33,000 daltons and an amino acid sequence essentally asdepicted in SEQ ID NO:37.
 5. The delta-endotoxin of claim 4 in which thedelta-endotoxin is obtained from Bacillus thuringiensis EMCC0075 havingthe identifying characteristics NRRL B-21019, or a spore or mutantthereof which have substantially the same properties as Bacillusthuringiensis EMCC0075 or Bacillus thuringiensis EMCC0076 having theidentifying characteristics of NRRL B-21020, or a spore or mutantthereof which have substantially the same properties as Bacillusthuringiensis EMCC0076.
 6. A delta-endotoxin having a molecular weightof about 33,000 daltons and an amino acid sequence essentially asdepicted in SEQ ID NO:38.
 7. The delta-endotoxin of claim 6 in which thedelta-endotoxin is obtained from Bacillus thuringiensis EMCC0075 havingthe identifying characteristics of NRRL B-21019, or a spore or mutantthereof which have substantially the same properties as Bacillusthuringiensis EMCC0075 or Bacillus thuringiensis EMCC0076 having theidentifying characteristics of NRRL B-21020, or a spore or mutantthereof which have substantially the same properties as Bacillusthuringiensis EMCCO0076.
 8. A nucleic acid fragment containing a nucleicacid sequence encoding the delta-endotoxin of claim 4 or a portion ofsaid delta-endotoxin having insecticidal activity against an insect pestof the order Lepidoptera.
 9. A nucleic acid fragment containing anucleic acid sequence encoding the delta-endotoxin or claim 6 orfragment thereof encoding a portion of said delta-endotoxin havinginsecticidal activity against an insect pest of the order Lepidoptera.10. A nucleic acid fragment containing a nucleic acid sequenceessentially as depicted in SEQ ID NO:39.
 11. A nucleic acid fragmentcontaining a nucleic acid sequence essentially as depicted in SEQ IDNO:40.
 12. A nucleic acid fragment containing a nucleic acid sequenceessentially as depicted in SEQ ID NO:41.
 13. A nucleic acid fragmentcontaining a nucleic acid sequence essentially as depicted in SEQ IDNO:44.
 14. A nucleic acid fragment containing a nucleic acid sequenceessentially as depicted in SEQ ID NO:45.
 15. A DNA construct comprisingthe nucleic acid fragment of claim
 8. 16. A DNA construct comprising thenucleic acid fragment of claim
 9. 17. A DNA construct comprising thenucleic acid fragment of claim
 10. 18. A DNA construct comprising (thenucleic acid fragment of claim
 11. 19. A DNA construct comprising thenucleic acid fragment of claim
 12. 20. A DNA construct comprising thenucleic acid fragment of claim
 13. 21. A DNA construct comprising thenucleic acid fragment of claim
 14. 22. A recombinant DNA vectorcomprising (a) the DNA construct of claim 15; (b) a promoter operablylinked to the DNA sequence of (a); and (c) a selectable marker.
 23. Arecombinant DNA vector comprising (a) the DNA construct of claim 16; (b)a promoter operably linked to the DNA sequence of (a); and (c) aselectable marker.
 24. A recombinant DNA vector comprising (a) the DNAconstruct of claim 17; (b) a promoter operably linked to the DNAsequence of (a); and (c) a selectable marker.
 25. A recombinant DNAvector comprising (a) the DNA construct of claim 18; (b) a promoteroperably linked to the DNA sequence of (a); and (c) a selectable marker.26. A recombinant DNA vector comprising (a) the DNA construct of claim19; (b) a promoter operably linked to the DNA sequence of (a); and (c) aselectable marker.
 27. A recombinant DNA vector comprising (a) the DNAconstruct of claim 20; (b) a promoter operably linked to the DNAsequence of (a); and (c) a selectable marker.
 28. A recombinant DNAvector comprising (a) the DNA construct of claim 21; (b) a promoteroperably linked to the DNA sequence of (a); and (c) a selectable marker.29. A host cell comprising (a) heterologous encoding the delta-endotoxinof claim 4 or a portion of said delta-endotoxin having insecticidalactivity against an insect pest of the order Lepidoptera.
 30. A hostcell comprising a heterologous nucleic acid containing a nucleic acidsequence encoding the delta-endotoxin of claim 6 or a portion of saiddelta-endotoxin having insecticidal activity against an insect pest ofthe order Lepidoptera.
 31. A host cell comprising the DNA construct ofclaim
 10. 32. A host cell comprising the DNA construct of claim
 11. 33.A host cell comprising the DNA construct of claim
 12. 34. A host cellcomprising the DNA construct of claim
 13. 35. A host cell comprising theDNA construct of claim
 14. 36. An insecticidal composition comprisingthe biologically pure Bacillus thuringiensis strain of claim 1 inassociation with an insecticidal carrier.
 37. An insecticidalcomposition comprising a delta-endotoxin having a molecular weight ofabout 33,000 daltons and an amino acid sequence essentially as depictedin SEQ ID NO:37 and a delta-endotoxin having a molecular weight of about33,000 daltons and an amino acid sequence essentially as depicted in SEQID NO:38 in association with an insecticidal carrier.
 38. Theinsecticidal composition of claim 37 in which the insecticidalcomposition further comprises spores of said biologically pure Bacillusthuringiensis strain.
 39. The insecticidal composition of claim 38 whichfurther comprises at least two delta-endotoxins having a molecularweight of about 130,000 and activity against an insect pest of the orderLepidoptera.
 40. A method for controlling an insect pest of the orderLepidoptera or Coleoptera comprising exposing the pest to aninsect-controlling effective amount of an insecticidal composition ofclaim
 37. 41. A method for controlling an insect pest of the orderLepidoptera comprising exposing the pest to an insect-controllingeffective amount of an insecticidal composition of claim 38.